The recommended ratio of medium volume to surface area for monolayer cell cultures is 0.2-0.3 ml/cm2 *. The upper limit is set by gaseous diffusion through the liquid; the optimum ratio depends on the oxygen requirement of the cells. Cells with a high oxygen requirement grow better in shallow medium (~2mm), those with a low requirement grow better in deep medium (~5mm). Exceeding a medium depth of 5mm can hinder gas exchange and impair growth.

*96WP requires a ratio of 0.3-0.4 ml/cm2 to overcome surface tension's effect on cell distribution and growth in the wells

*Volumes used in tissue culture flasks are limited by the locations of the neck